The present invention concerns the field of molecular genetics and medicine. Particularly, it concerns genes encoding a protein that is involved in homologous recombination and the repair of damaged genomic DNA in mammalian cells. Specifically the invention concerns the gene encoding a mammalian ATP-dependent homologous pairing protein; methods of using the gene to effect gene therapy; methods of using the gene and fragments of the gene to classify a mammalian tissue for medical purposes; and transgenic mice having had one or both alleles of the gene made inoperative. More specifically, the gene of the present invention is the human and murine homologs of the gene termed REC2 previously isolated from the fungus Ustilago maydis. 
During the life of every organism the DNA of its cells is constantly subjected to chemical and physical events that cause alterations in its structure, i.e., potential mutations. These potential mutations are recognized by DNA repair enzymes found in the cell because of the mismatch between the strands of the DNA. To prevent the deleterious effects that would occur if these potential mutations became fixed, all organisms have a variety of mechanisms to repair DNA mismatches. In addition, higher animals have evolved mechanisms whereby cells having highly damaged DNA, undergo a process of programmed death (xe2x80x9capoptosisxe2x80x9d).
The association between defects in the DNA mismatch repair and apoptosis inducing pathways and the development, progression and response to treatment of oncologic disease is widely recognized, if incompletely understood, by medical scientists. Chung, D. C. and Rustgi, A. K., 1995, Gastroenterology 109:1685-99; Lowe, S. W., et al., 1994, Science 266:807-10. Therefore, there is a continuing need to identify and clone the genes that encode proteins involved in DNA repair and DNA mismatch monitoring.
Studies with bacteria, fungi and yeast have identified three genetically defined groups of genes involved in mismatch repair processes. The groups are termed, respectively, excision repair group, the error prone repair group and recombination repair group. Mutants in a gene of each group results in a characteristic phenotype. Mutants in the recombination repair group in yeast result in a phenotype having extreme sensitivity to ionizing radiation, a sporulation deficiency, and decreased or absent mitotic recombination. Petes, T. D., et al., 1991, in Broach, J. R., et al., eds., The Molecular Biology of the Yeast Saccharomyces, pp. 407-522 (Cold Spring Harbor Press, 1991).
Several phylogenetically related genes have been identified in the recombination repair group: recA, in E. Coli, Radding, C. M., 1989, Biochim. Biophys. Acta 1008:131-145; RAD51 in S. cerevisiae, Shinohara, A., 1992, Cell 69:457-470, Aboussekhra, A. R., et al., 1992, Mol. Cell. Biol. 12:3224-3234, Basile, G., et al., 1992, Mol. Cell. Biol. 12:3235-3246; RAD57 in S. cerevisiae, Gene 105:139-140; REC2 in U. maydis, Bauchwitz, R., and Holloman, W. K., 1990, Gene 96:285-288, Rubin, B. P., et al., 1994, Mol. Cell. Biol. 14:6287-6296. A third S. cerevisiaegene DMC1, is related to recA, although mutants of DMC1 show defects in cell-cycle progression, recombination and meiosis, but not in recombination repair.
The phenotype of REC2 defective U. maydis mutants is characterized by extreme sensitivity to ionizing radiation, defective mitotic recombination and interplasmid recombination, and an inability to complete meiosis. Holliday, R., 1967, Mutational Research 4:275-288. UmREC2, the REC2 gene product of U. maydis, has been extensively studied. It is a 781 amino acid ATPase that, in the presence of ATP, catalyzes the pairing of homologous DNA strands in a wide variety of circumstances, e.g., UmREC2 catalyzes the formation of duplex DNA from denatured strands, strand exchange between duplex and single stranded homologous DNA and the formation of a nuclease resistant complex between identical strands. Kmiec, E. B., et al., 1994, Mol. Cell. Biol. 14:7163-7172. UmREC2 is unique in that it is the only eukaryotic ATPase that forms homolog pairs, an activity it shares with the E. coli enzyme recA.
U.S. Pat. No. 5,780,296 filed Jan. 17, 1995, by W. K. Holloman and E. B. Kmiec discloses REC2 from U. maydis, methods of producing recombinant UmREC2 and methods of its use. Prior to the date of the present invention a fragment of human REC2 cDNA was available from the IMAGE consortium, Lawrence Livermore National Laboratories, as plasmid p153195. Approximately 400 bp of the sequence of p153195 had been made publicly available on dbEST database.
The scientific publication entitled: Isolation of Human and Mouse Genes Based on Homology to REC2, July 1997, Proc. Natl. Acad. Sci. 94, 7417-7422 by Michael C. Rice et al., discloses the sequences of murine and human Rec2, of the human REC2 cDNA. and discloses that irradiation increases the level of hsREC2 transcripts in primary human foreskin fibroblasts.
The invention provides nucleic acids encoding mammalian ATP-dependent homologous pairing proteins (a xe2x80x9cmammalian recombinasexe2x80x9d) particularly, the human and murine ATP-dependent homologous pairing protein (hsREC2 and muREC2, respectively). The invention additionally provides DNA clones containing a copy of the mRNA encoding a mammalian recombinase (an xe2x80x9cmREC cDNAxe2x80x9d) and DNA clones containing a copy of the genomic DNA containing an mREC gene or fragments thereof. In further embodiments, the invention concerns a nucleic acid comprising an mREC cDNA linked to a heterologous promoter, i.e., a promoter other than a mammalian recombinase promoter, so that a mammalian recombinase can be expressed or over-expressed in insect and mammalian cells and in bacteria. In one embodiment, the heterologous promoter is the polyhedrin promoter from the baculovirus Autographica californica and the invention provides for an isolated and purified mammalian recombinase, particularly isolated and purified hsREC2.
The invention provides several utilities of said nucleic acids and isolated and purified proteins. In the area of gene therapy and the construction of transgenic animals, the invention provides a method of enhancing homologous recombination between an exogenous nucleic acid and the genome of a cell by introducing a plasmid that expresses an mammalian recombinase into the cell, which method can be used to repair a genetic defect and thereby cure a genetic disease. Alternatively, for the construction of transgenic animals the invention provides a method of enhancing homologous recombination between an exogenous nucleic acid and the genome of a cell by introducing purified mammalian recombinase into the cell. Alternatively, the invention provides for the construction of a transgenic animal, such as a mouse, having a transgenic mammalian recombinase gene operably linked to a strong promoter so that the recombinase is over expressed in some or all tissues. Such transgenic animals are highly adapted to undergo homologous recombination.
The invention additionally provides two methods of classifying a sample of human tissue for diagnosis and prognosis: by determining whether the cells of the sample contains two, one or no copies of hsREC2; and by determining whether the copies of hsREC2 that said cells contain are mutated. For the purpose of diagnosis and classification of tissue samples the invention, firstly, provides paired oligonucleotides that are suitable for the PCR amplification of fragments of hsREC2 and, secondly, identifies a microsatellite DNA sequence, D14S258, that is closely linked to hsREC2.
The invention further provides a transgenic mouse having one or both alleles of muREC2 interrupted and thereby inactivated. The resultant transgenic animals, termed heterozygous and homozygous REC2-knock out mice, respectively, are susceptible to tumorigenesis by chemical carcinogens. REC2-knock-out mice can be used to determine whether their is a significant risk of carcinogenesis associated with a chemical or a process of interest. The reduced level or absence of muREC2 makes REC2-knock-out mice a more sensitive test animal than wild-type.
The invention further provies a method of sensitizing target cells to DNA damage, such as from xcex3- or UV irradiation or from cytotoxic agents commonly used in oncologic therapy, which comprises causing the expression of high levels of recombinase in the target cell. The expression of such levels causes the cells to more readily undergo apoptosis in response to DNA damage. The invention yet further provides the REC2 promoter a mammalian promoter that is induceable by irradiation or other DNA damaging agents.